Antiviral Activity of Benzavir-2 against Emerging Flaviviruses

Gwon, Yong-Dae; Strand, Mårten; Lindqvist, Richard; Nilsson, Emma; Saleeb, Michael; Elofsson, Mikael; Överby, Anna K.; Evander, Magnus

To obtain insight into the mode-of-action of benzavir-2, a time-of-addition experiment was performed. Vero B4 cells were infected with rZIKV-ZsGreen and benzavir-2 was added at different time points; pre-treatment (−2 to 0 h) for every second hour in 2 h pulses (0-2 h, 2-4 h, 4-6 h, or 6-8 h) and for 8 h (0-8 h) . Based on the growth kinetics of rZIKV-ZsGreen and the sensitivity of the ZsGreen detection in the fluorescent intensity assay, the analysis of virus infection was performed 24 h post-infection, and a 2-h treatment scheme with benzavir-2 at a relatively high concentration (10 µM) was selected ( Figure 5A ). When benzavir-2 was added at 2-4 h post-infection (hpi), the inhibitory effect was most pronounced, while the binding and entry steps (0-2 h) were less affected. Addition of benzavir-2 at later time-points post-infection also showed less inhibition. Although pre-incubation of the cells with benzavir-2 prior to infection (−2 h) had an inhibitory effect; it was much lower compared to the addition of benzavir-2 in the early phase (2-4 h) of infection ( Figure 5B ).